1

Closing in on DiGeorge: HumanSyndromes and Mouse Models

The DiGeorge Syndrome (DGS)

• Cardiac outflow tract & septal defects• Thymus and parathryoid hypoplasia• Laryngeotrachial anomalies• Craniofacial anomalies/facial dysmorphogenesis• Cleft palate• Micrognathia• Low-set, abnormal ears• Mental retardation

The DiGeorge/VelocardiofacialSyndrome (DGS/VCFS)

• Chr22 deletions• Overlapping clinical features with

Velocardiofacial syndrome, conotruncal facesyndrome

• CATCH 22: Cardiac, T-cell deficit, Clefting, Hypocalcemia, Chromosome 22

(it lived up to its namesake!)

Etiology of DGS

• Variable size deletions 1.5-3MB• No common region of overlap• Contiguous gene syndrome?• Single gene haploinsufficiency?

• Pharyngeal arch and pouch developmental defects• Neural crest defects

Developmental

Genetic

Regions of Conserved Synteny Human Chromosome 22

MouseChromosome 16

MouseChromosome Synteny

61610 51110 8

15

22q1122q12

22q13

Mouse Chromosome 16DGS syntenicregion genes

Deletionmodels

Candidate gene approach

Znf74Dgcr3IddTsk2Tsk1Es2GsclCtpDgcr6ProdhRanbp1Htf9cT10ArvcfComtTrxR2Wdvcf

Tbx1Gp1bCdcrel1TmvcfCdc45f2Ufd1lNivcfHira

+ ++ +

--

- Crkl

Es2Gscl

Tbx1Gp1bComtUfd1lHira

+=cardiovascular defect

2

What is Tbx1Is Tbx1 the key?

The T-box Transcription Factor Gene Family

• 17 genes in mouse and human• Conserved DNA binding domain - the T-domain• Bind sequence-specific DNA as dimers• Affect transcription of target genes• Conserved developmental functions• Tbx1 maps to DGS region

T-domain dimer bound to DNAMuller & Herrmann (1997) Nature 389:884

T-box gene mutations in human and mouse

• TBX3 - ulnar-mammarysyndrome

• TBX4 - small patellasyndrome

• TBX5 - Holt-Oramsyndrome

• TBX19 - pituitary deficiencyof ACTH

• TBX22 - cleft palate withankyloglossia

• Tbx2 – heart, limbs• Tbx3 – limbs, mammary, yolk sac• Tbx4 – hindlimb, allantois• Tbx5 – heart, forelimb• Tbx6 – paraxial mesoderm• T – posterior mesoderm, notochord• Tbr1 – brain• Eomes – trophectoderm, mesoderm• Tbet – T cells• Tbx15 – D/V patterning• Tbx18 – somite patterning• Tbx20 – heart patterning

Human Mouse

Chromosomal Locations of T-box Genes

0

100

40

60

20

80

0

40

60

20

80

2 3 5 7

9 11 16 17 19

Tbx10TTbx1

Tbx2,Tbx4

eomes

Tbx6Tbx3,Tbx5

Tbx15Tbr1

a

fi

Lmx1b

Egf

Fgf2

jg

kitIgf1r

Fgfr2

du

Bmp5 oe

Hoxb

Dgscgsc

tfFgf8ru

Tbx1 Expression at midgestation Tbx1 Expression at midgestation

3

Tbx1 Targeted Mutagenesis

• Heterozygotes are viable and fertile• Homozygotes die at birth

+/-+/+

E11.5

Aortic Arch Artery Defect in Tbx1Heterozygous Embryos

neonates

Ear Defect in Tbx1 HomozygousMutant Newborns

Craniofacial Abnormalities in Tbx1Mutants

+/+

-/-

Cleft Palate in Tbx1 Mutant Mice

p

m

-/-+/+

Glandular and Heart Abnormalitiesin Tbx1 Mutant Mice

4

+/-+/+

-/- E11.5

Aortic Arch Abnormalities

e11.5

Cardiovascular Defects in Tbx1Mutant Mice

+/+ -/-

Tbx1+/-

Tbx1-/-

E12.5MyoD

Failure of branchiomeric myogenesis inTbx1-/- embryos

Tbx1+/- Tbx1-/-

E14.5

FGF10 enhancer trap

Craniofacial (branchiomeric)musculature not specified in Tbx1 mutant

mice

Otic Vesicle, Pharyngeal Archand Pouch Defects

E9.5

+/+ -/-

Major Embryonic Derivativesof Tbx1 Expressing Tissues

• Otic vesicle – inner ear• Pharyngeal arches – craniofacial bones,

neck cartilage, ears,musculature

• Pharyngeal pouches – thymus, parathyroid• Pharyngeal arch arteries – aorta• (Neural crest – cardiac septum)

5

Tbx1 mutant mice have many features ofDiGeorge syndrome

Low set, abnormal earsAbnormal aortic arches

Hypoplastic thymus Micrognathia Cleft palateSeptal defects

+/+ +/-

+/+ -/- +/+

-/-

+/+ -/-

DGS/VCFS Tbx1 Mutant

• Cardiac outflow tract &septal defects

• Thymus & parathryoidhypoplasia

• Laryngeotrachialanomalies

• Craniofacial anomalies• Cleft palate• Micrognathia• Low-set, abnormal ears• Muscle hypotonia

• Cardiac outflow tract &septal defects

• Thymus & parathryoidhypoplasia

• Laryngeotrachialanomalies

• Craniofacial anomalies• Cleft palate• Micrognathia• Low-set, abnormal ears• Myogenesis defects

Salivary gland defects in Tbx1mutant mice

sublingual and submaxillary salivary glands missing or reduced

Is TBX1 the only gene involvedin DiGeorge syndrome?

• Mouse haploinsufficiency does not recapitulate DGS• Not all DGS deletions include TBX1• Most non-deletion DGS patients do not have TBX1

mutations• However, several non-deletion pedigrees do have

TBX1 mutations

Issues to be resolved

Is TBX1 the only gene involvedin DiGeorge syndrome?

• Deletion of regulatory elements?• Linked modifier loci?• Chromatin conformational effects?• Linked genes affecting the same tissues?• Exacerbation of haploinsufficiency?

Genetic interactions with linkedgenes

• Crkl gene in 22q11• Sometimes deleted in DGS• Expressed in neural crest• Exacerbated cardiac phenotype in Crkl;

Tbx1 compound mutants

6

Genetic interaction Crkl

pharyngeal pouch development as visualized by Pax1 in situ hybridization

Closing in on DiGeorge: HumanSyndromes and Mouse Models

AcknowledgementsColumbia University

Loydie Jerome-MajewskaDebbie ChapmanJeremy Gibson-BrownSarah HancockRobert Kelly

Collaborators Lee Silver Debbie Guris Akira Imamoto